Fiber-optic assay apparatus based on phase-shift interferometry

ABSTRACT

Apparatus and method for detecting the presence or amount or rate of binding of an analyte in a sample solution is disclosed. The apparatus includes an optical assembly having first and second reflecting surfaces separated by a distance “d” greater than 50 nm, where the first surface is formed by a layer of analyte-binding molecules, and a light source for directing a beam of light onto said first and second reflecting surface. A detector in the apparatus operates to detect a change in the thickness of the first reflecting layer resulting from binding of analyte to the analyte-binding molecules, when the assembly is placed in the solution of analyte, by detecting a shift in phase of light waves reflected from the first and second surfaces.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/518,068, filed Nov. 6, 2003, and U.S. Provisional Application No.60/558,381, filed Mar. 31, 2004 the entire disclosures of which arehereby incorporated by reference in their entirety for all purposes.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not applicable.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to an apparatus and method for detectingthe presence, amount, or rate of binding of one or more analytes in asample, and in particular, to apparatus and method based on fiber opticinterferometry.

2. Description of the Related Art

Diagnostic tests based on a binding event between members of ananalyte-anti-analyte binding pair are widely used in medical,veterinary, agricultural and research applications. Typically, suchmethods are employed to detect the presence or amount or an analyte in asample, and/or the rate of binding of the analyte to the anti-analyte.Typical analyte-anti-analyte pairs include complementary strands ofnucleic acids, antigen-antibody pairs, and receptor-receptor bindingagent, where the analyte can be either member of the pair, and theanti-analyte molecule, the opposite member.

Diagnostics methods of this type often employ a solid surface havingimmobilized anti-analyte molecules to which sample analyte moleculeswill bind specifically and with high affinity at a defined detectionzone. In this type of assay, known as a solid-phase assay, the solidsurface is exposed to the sample under conditions that promote analytebinding to immobilized anti-analyte molecules. The binding event can bedetected directly, e.g., by a change in the mass, reflectivity,thickness, color or other characteristic indicative of a binding event.Where the analyte is pre-labeled, e.g., with a chromophore, orfluorescent or radiolabel, the binding event is detectable by thepresence and/or amount of detectable label at the detection zone.Alternatively, the analyte can be labeled after it is bound at thedetection zone, e.g., with a secondary, fluorescent-labeled anti-analyteantibody.

Co-owned U.S. Pat. No. 5,804,453, (the '453 patent) which isincorporated herein by reference, discloses a fiber-optic interferometerassay device designed to detect analyte binding to a fiber-optic endsurface. Analyte detection is based on a change in the thickness at theend surface of the optical fiber resulting from the binding of analytemolecules to the surface, with greater amount of analyte producing agreater thickness-related change in the interference signal. The changein interference signal is due to a phase shift between light reflectedfrom the end of the fiber and from the binding layer carried on thefiber end, as illustrated particularly in FIGS. 7a and 7b of the '453patent. The device is simple to operate and provides a rapid assaymethod for analyte detection.

Ideally, an interferometer assay device will yield readily observablechanges in spectral peak and valley (extrema) positions within the rangeof a conventional visible-light spectrometer, that is, in the visiblelight range between about 450-700 nm, such that relatively small opticalthickness changes at the fiber end can be detected as significantchanges in the spectral positions of interference wavelength peaks andvalleys. One limitation which has been observed with the devicedescribed in the '453 patent is the absence of readily identifiedwavelength spectral extrema over this spectral range.

The present invention is designed to overcome this limitation,preserving the advantages of speed and simplicity of theearlier-disclosed device, but significantly enhancing sensitivity andaccuracy. The present invention also provides a more convenientdisposable-head format, as well as a multi-analyte array format, e.g.,for gene-chip and protein-chip applications.

SUMMARY OF THE INVENTION

The invention includes, in one aspect, an apparatus for detecting ananalyte in a sample, including detecting the presence of analyte, theamount of analyte or the rate of association and/or dissociation ofanalyte to analyte-binding molecules. The apparatus includes an opticalelement with a proximal reflecting surface and a distal reflectingsurface separated by at least 50 nm. A beam of light from an opticalfiber is directed to and reflected from the two reflecting surfaces. Thereflected beams are coupled back into the optical fiber and interfere.The optical element also includes a layer of analyte binding moleculesthat is positioned so that the interference between the reflected beamsvaries as analyte binds to the layer of analyte binding molecules.

The change in interference can be caused by different physicalphenomenon. For example, analyte binding can cause a change in theoptical path length or in the physical distance between the tworeflecting surfaces. Alternately, analyte binding can cause a change inthe index or in the optical absorption of material located between thereflecting surfaces. Analyte binding can also cause the layer of analytebinding molecules to swell, resulting in a change in the interference.

In one particular design, the distal reflecting surface includes thelayer of analyte binding molecules. As analyte binds to the layer ofanalyte binding molecules, the optical path length or the physicaldistance between the two reflecting surfaces may increase, for example.In another aspect of the invention, a transparent solid material islocated between the reflecting surfaces and, optionally, the proximalreflecting surface includes a material with an index greater than thatof the transparent solid material. Alternately, an air gap may belocated between the reflecting surfaces. In yet another design, thedistal reflecting surface is positioned between the proximatelyreflecting surface and the layer of analyte binding molecules. Forexample, analyte binding may cause the layer of analyte bindingmolecules to swell, moving the distal reflecting surface closer to theproximal reflecting surface. In yet another design, the layer of analytebinding molecules is positioned between the two reflecting surfaces.Analyte binding may cause the layer to swell or to change its index,thus changing the interference between the two reflected beams.

In another aspect, the apparatus includes an optical assembly havingfirst and second reflecting surfaces separated by a distance “d” greaterthan 50 nm. The optical assembly is composed of a transparent opticalelement that can have a thickness defined between proximal and distalfaces of the element of at least 50 nm, preferably between 400-1,000 nm.The first reflecting surface is carried on the distal face of opticalelement, and is formed of a layer of analyte-binding molecules. Thesecond reflecting surface is formed by a coating of transparent materialhaving an index of refraction greater than that of the optical element.This coating can be formed of a Ta₂O₅ layer having a preferred thicknessof between 5 and 50 nm. The optical element can be SiO₂, and has athickness of between about 100-5,000 nm, preferably 400-1,000 nm.

Also included are a light source for directing a beam of light onto thefirst and second reflecting surfaces, and a detector unit that operatesto detect a change in the optical thickness of the first reflectinglayer resulting from binding of analyte to the analyte-bindingmolecules, when the assembly is placed in the solution of analyte. Theoptical thickness change at the first reflecting layer is related to ashift in a phase characteristic of the interference wave formed by thetwo light waves reflected from said first and second surfaces. Thisphase characteristic can be a shift in the spectral position(s) of oneor more peaks and valleys of the interference wave, or by a change inthe period of a full cycle of the wave.

The light source can include an optical fiber having a distal endadapted to be placed adjacent the second reflecting surface in theassembly, and the apparatus further includes an optical coupling fordirecting reflected light waves reflected from the assembly to thedetector.

In a first embodiment, the optical assembly is fixedly mounted on theoptical fiber, with the distal end of the optical fiber in contact withthe second reflecting surface. In a second embodiment, the opticalassembly further includes a second transparent optical element having anindex of refraction less than that of the second coating and a thicknessgreater than about 100 nm, where the coating of high index of refractionmaterial is sandwiched between the two transparent optical elements. Inthis latter embodiment, the assembly is removably attached to the distalend region of the fiber with a spacing of less than 100 nm or greaterthan 2 μm between the distal end of the fiber and the confronting faceof the second transparent optical element in the assembly.

For detecting multiple analytes, such as multiple nucleic acid species,the layer of analyte-binding molecules can be composed of an array ofdiscrete analyte-binding regions, such as single strands of nucleicacid. The regions are effective to bind different analytes. The opticalfiber includes a plurality of individual fibers, each aligned with oneof the regions, the detector includes a plurality of detection zones,and the optical coupling functions to couple each of the plurality offibers with one of the zones.

The analyte-binding molecules in the assembly can be, for example, (i)an anti-species antibody molecules, for use in screening hybridomalibraries for the presence of secreted antibody, (ii) antigen molecules,for use in detecting the presence of antibodies specific against thatantigen; (iii) protein molecules, for use in detecting the presence of abinding partner for that protein; (iv) protein molecules, for use indetecting the presence of multiple binding species capable of forming amulti-protein complex with the protein; or (v) single stranded nucleicacid molecules, for detecting the presence of nucleic acid bindingmolecules.

The detector can be a spectrometer for measuring reflected lightintensity over a selected range of wavelengths. Alternatively, or inaddition, the light source can include a plurality of light-emittingdiodes, each with a characteristic spectral frequency, and the detectorfunctions to record light intensity of reflected light at each of thedifferent LED frequencies. In still another embodiment, the light sourceincludes a white-light source and the detector is designed to recordlight intensity of reflected light at each of a plurality of differentwavelengths.

In another aspect, the invention includes a method for detecting thepresence or amount of an analyte in a sample solution. The methodinvolves reacting the sample solution with a first reflecting surfaceformed by a layer of analyte-binding molecules carried on the distalsurface of a transparent optical element having a thickness of at least50 nm, thereby to increase the thickness of the first reflecting layerby the binding of analyte to the analyte-binding molecules in the layer.The change in thickness of the first reflecting layer is measured bydetecting a shift in a phase characteristic of the interference waveformed by the two light waves reflected from the first layer and from asecond reflecting layer that is formed on the opposite, proximal surfaceof the optical element and which has an index of refraction greater thanthat of the optical element.

The detecting step can include directing light from an optical fiberonto the two reflecting surfaces, and directing reflected light from thetwo surfaces onto a detector through an optical coupling. The detectorcan be a spectrometer, where the detecting includes measuring a shift inthe spectral position of one or more of the interference extremaproduced by the two reflecting lightwaves.

Where the method is used for measuring the rate of association ofanalyte to the second layer, the reacting step can be carried out untila near-maximum increase in thickness of the first reflecting layer isobserved. Where the method is used for measuring the rate ofdissociation of analyte to the second layer, the reacting steps caninclude immersing the second layer in a dissociation buffer for a periodof time until a decrease in thickness of the first reflecting layer isobserved. Where the method is used for measuring the amount of analytepresent in the sample, the detecting is carried out over a periodsufficient to measure the thickness of the first reflecting layer at aplurality of different time points.

Where the method is used measuring one or more of a plurality ofanalytes in a sample, the first reflecting layer is composed of an arrayof discrete analyte-binding regions, the different regions beingeffective to bind different analytes, and the detecting is effective todetect a change in the thickness of each of the regions resulting frombinding of analyte to the analyte-binding molecules.

These and other objects and features of the present invention willbecome more fully apparent when the following detailed description ofthe invention is read in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING

These and other features, aspects, and advantages of the presentinvention will become better understood with regard to the followingdescription, and accompanying drawings, where:

FIG. 1 shows the basic system setup for the bioprobe and its apparatus;

FIG. 2 shows an optical assembly formed accordance to one embodiment ofthe invention;

FIGS. 3A and 3B show a portion of an interference wave over 7 peak andvalley orders (3A), and over in a visible portion of the spectrum (3B);

FIG. 4 shows an optical assembly constructed according to anotherembodiment of the invention;

FIG. 5 shows a disposable multi-analyte optical assembly having ananalyte-binding array and constructed according to another embodiment ofthe invention;

FIG. 6 shows a sequential binding of three molecules;

FIG. 7 shows on and off curves generated from the association anddissociation of antibodies;

FIG. 8 shows the curves of two antibodies binding to their antigen atdifferent concentrations;

FIG. 9 shows immobilization of bis amino PEG (MW 3300) specificallythrough an amide bond formation. The PEG (MW 8000) is used as a negativecontrol to monitor non-specific binding of the PEG polymer; and

FIG. 10 shows a small molecule binding to a large molecule, negativecontrols and the base line measurement.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

Terms used in the claims and specification are to be construed inaccordance with their usual meaning as understood by one skilled in theart except and as defined as set forth below. Numeric ranges recited inthe claims and specification are to be construed as including the limitsbounding the recited ranges.

The term “in vivo” refers to processes that occur in a living organism.

An “analyte-binding” molecule refers to any molecule capable ofparticipating in a specific binding reaction with an analyte molecule.Examples include but are not limited to, e.g., antibody-antigen bindingreactions, and nucleic acid hybridization reactions.

A “specific binding reaction” refers to a binding reaction that issaturable, usually reversible, and that can be competed with an excessof one of the reactants. Specific binding reactions are characterized bycomplementarity of shape, charge, and other binding determinants asbetween the participants in the specific binding reaction.

An “antibody” refers to an immunoglobulin molecule having two heavychains and two light chains prepared by any method known in the art orlater developed and includes polyclonal antibodies such as thoseproduced by inoculating a mammal such as a goat, mouse, rabbit, etc.with an immunogen, as well as monoclonal antibodies produced using thewell-known Kohler Milstein hybridoma fusion technique. The term includesantibodies produced using genetic engineering methods such as thoseemploying, e.g., SCID mice reconstituted with human immunoglobulingenes, as well as antibodies that have been humanized using art-knownresurfacing techniques.

An “antibody fragment” refers to a fragment of an antibody moleculeproduced by chemical cleavage or genetic engineering techniques, as wellas to single chain variable fragments (SCFvs) such as those producedusing combinatorial genetic libraries and phage display technologies.Antibody fragments used in accordance with the present invention usuallyretain the ability to bind their cognate antigen and so include variablesequences and antigen combining sites.

A “small molecule” refers to an organic compound having a molecularweight less than about 500 daltons. Small molecules are useful startingmaterials for screening to identify drug lead compounds that then can beoptimized through traditional medicinal chemistry, structure activityrelationship studies to create new drugs. Small molecule drug compoundshave the benefit of usually being orally bioavailable. Examples of smallmolecules include compounds listed in the following databases: MDL/ACD(http://www.mdli.com/), MDL/MDDR (http://www.mdli.com/), SPECS(http://www.specs.net/), the China Natural Product Database (CNPD)(http://www.neotrident.com/), and the compound sample database of theNational Center for Drug Screening (http://www.screen.org.cn/).

Abbreviations used in this application include the following: “ss”refers to single-stranded; “SNP” refers to single nucleotidepolymorphism; “PBS” refers to phosphate buffered saline (0.01 Mphosphate buffer, 0.0027 M potassium chloride and 0.137 M sodiumchloride, pH 7.4); “NHS” refers to N-hydroxysuccinimide; “MW” refers tomolecular weight; “Sulfo-SMCC” refers to sulfosuccinimidyl4-[N-maleimidomethyl]cyclohexane-1-carboxylate.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an” and “the” include plural referentsunless the context clearly dictates otherwise.

Advantages and Utility

The advantages and utility of the invention are illustrated by referenceto the Figures and Examples as described in greater detail below. Theseinclude the ability to monitor in real time analyte binding reactionswithout the use of labels, diminishing cost and potential toxicity. Afurther advantage includes the ability to practice the method usingvisible wavelength light sources. Yet other advantages are provided bythe fiber optic nature of the detector tip that allows binding reactionsto be monitored in very small sample volumes, including in “in vitro”spaces, and to bundle fibers to carry out highly multiplexed analyses ofbinding reactions.

FIG. 1 shows, in schematic view, an interferometer apparatus 20constructed in accordance with the invention. In its most basicelements, the apparatus includes a light source 22, an optical assembly26 that functions as a sensing element or detector tip and that will bedetailed further with respect to FIGS. 2, 4 and 5 below, and a detectorunit 28 for detecting interference signals produced by interfering lightwaves reflected from the optical assembly 26.

Light from source 22 is directed onto the optical assembly 26, andreflected back to the detector through an optical coupling assemblyindicated by dashed lines at 30. In a preferred embodiment, the couplingassembly includes a first optical waveguide or fiber 32 extending fromthe light source to the optical assembly, a second optical waveguide orfiber 34 which carries reflected light from the optical assembly to thedetector, and an optical coupler 36 which optically couples fibers 32,34. Suitable optical fiber and coupling components are detailed in theabove-cited '453 patent. One exemplary coupler is commercially availablefrom many vendors including Ocean Optics (Dunedin, Fla.).

Alternatively, the coupling assembling can include a lens systemconstructed to focus a light beam onto the upper surface of the opticalassembly and to direct reflected interfering light from the opticalassembly to the detector. The latter system would not require opticalfibers, but would impose relatively stringent requirements on thepositioning of the lens elements used for the optical coupling.

The light source in the apparatus can be a white light source, such as alight emitting diode (LED) which produces light over a broad spectrum,e.g., 400 nm or less to 700 nm or greater, typically over a spectralrange of at least 100 nm. Alternatively, the light source can be aplurality of sources each having a different characteristic wavelength,such as LEDs designed for light emission at different selectedwavelengths in the visible light range. The same function can beachieved by a single light source, e.g., white light source, withsuitable filters for directing light with different selected wavelengthsonto the optical assembly.

The detector is preferably a spectrometer, such as charge-coupled device(CCD), capable of recording the spectrum of the reflected interferinglight from the optical assembly. Alternatively, where the light sourceoperates to direct different selected wavelengths onto the opticalassembly, the detector can be a simple photodetector for recording lightintensity at each of the different irradiating wavelengths. In stillanother embodiment, the detector can include a plurality of filterswhich allows detection of light intensity, e.g., from a white-lightsource, at each of a plurality of selected wavelengths of theinterference reflectance wave. Exemplary light source and detectorconfigurations are described in the above-cited '453 patent,particularly with respect to FIGS. 8 and 10 of that patent, and it willbe understood that these configurations are suitable for use in thepresent invention.

FIG. 2 shows an optical assembly 26 constructed in accordance with oneembodiment of the invention, and an adjoining portion of the distal endregion of an optical fiber 32 to which the optical assembly is fixedlyattached. As seen, the assembly 26 includes a transparent opticalelement 38 having first and second reflecting surfaces 42, 40 formed onits lower (distal) and upper (proximal) end faces, respectively.According to an important feature of the invention, the thickness “d” ofthe optical element between its distal and proximal surfaces, i.e.,between the two reflecting surfaces, is at least 50 nm, and preferablyat least 100 nm. An exemplary thickness is between about 100-5,000 nm,preferably 400-1,000 nm. The first reflecting surface 42 is formed of alayer of analyte-binding molecules, such as molecules 44, which areeffective to bind analyte molecules 46 specifically and with highaffinity. That is, the analyte and anti-analyte molecules are oppositemembers of a binding pair of the type described above, which caninclude, without limitations, antigen-antibody pairs, complementarynucleic acids, and receptor-binding agent pairs.

The index of refraction of the optical element is preferably similar tothat of the first reflecting surface, so that reflection from the lowerdistal end of the end optical assembly occurs predominantly from thelayer formed by the analyte-binding molecules, rather than from theinterface between the optical element and the analyte-binding molecules.Similarly, as analyte molecules bind to the lower layer of the opticalassembly, light reflection form the lower end of the assembly occurspredominantly from the layer formed by the analyte-binding molecules andbound analyte, rather than from the interface region. One exemplarymaterial forming the optical element is SiO₂, e.g., a high-qualityquality glass having an index of refraction of about 1.4-1.5. Theoptical element can also be formed of a transparent polymer, such aspolystyrene or polyethylene, having an index of refraction preferably inthe 1.3-1.8 range.

The second reflecting surface in the optical assembly formed as a layerof transparent material having an index of refraction that issubstantially higher than that of the optical element, such that thislayer functions to reflect a portion of the light directed onto theoptical assembly. Preferably, the second layer has a refractive indexgreater than 1.8. One exemplary material for the second layer is Ta₂O₅with refractive index equal to 2.1. The layer is typically formed on theoptical element by a conventional vapor deposition coating or layeringprocess, to a layer thickness of less than 50 nm, typically between 5and 30 nm.

The thickness of the first (analyte-binding) layer is designed tooptimize the overall sensitivity based on specific hardware and opticalcomponents. Conventional immobilization chemistries are used inchemically, e.g., covalently, attaching a layer of analyte-bindingmolecules to the lower surface of the optical element. For example, avariety of bifunctional reagents containing a siloxane group forchemical attachment to SiO₂, and an hydroxyl, amine, carboxyl or otherreaction group for attachment of biological molecules, such as proteins(e.g., antigens, antibodies), or nucleic acids. It is also well known toetch or otherwise treat glass a glass surface to increase the density ofhydroxyl groups by which analyte-binding molecules can be bound. Wherethe optical element is formed of a polymer, such as polystyrene, avariety of methods are available for exposing availablechemically-active surface groups, such as amine, hydroxyl, and carboxylgroups.

The analyte-binding layer is preferably formed under conditions in whichthe distal surface of the optical element is densely coated, so thatbinding of analyte molecules to the layer forces a change in thethickness of the layer, rather than filling in the layer. Theanalyte-binding layer can be either a monolayer or a multi-layer matrix.

The measurement of the presence, concentration, and/or binding rate ofanalyte to the optical assembly is enabled by the interference ofreflected light beams from the two reflecting surfaces in the opticalassembly. Specifically, as analyte molecules attach to or detach fromthe surface, the average thickness of the first reflecting layer changesaccordingly. Because the thickness of all other layers remains the same,the interference wave formed by the light waves reflected from the twosurfaces is phase shifted in accordance with this thickness change.

Assume that there are two reflected beams: The first beam is reflectedfrom the first surface, which is the distal end interface betweenanalyte-binding molecules and bound analyte and the surrounding medium;and the second beam is reflected from the second surface, which is theproximal interface between the optical element (the first layer) and thehigh-index of refraction layer (the second layer). The overallwavelength-dependent intensity of the interference wave is:

$I = {I_{1} + I_{2} + {2\sqrt{I_{1}I_{2}}{\cos\left( \frac{2\pi\;\Delta}{\lambda} \right)}}}$

where I is the intensity, I₁ and I₂ are the intensity of twointerference beams, Δ is the optical path difference, and λ is thewavelength.

When (2πΔ/λ)=Nπ, the curve is at its peak or valley if N is an integer0, 1, 2, . . . .

The thickness of the first layer d=Δ/2n. Therefore, λ=4nd/N at peaks orvalleys (extrema).

For the first several values of N, i.e., 0, 1, 2, . . . 7, and assuminga d of 770 nm, the equation gives:

-   -   N=0: λ=∞ (peak)    -   N=1: λ=4nd=4,496.80 nm (Valley)    -   N=2: λ=2nd=2,248.40 nm (Peak)    -   N=3: λ=4nd/3=1,498.9 nm (Valley)    -   N=4: λ=nd=1,124.20 nm (Peak)    -   N=5: λ=4nd/5=899.36 nm (Valley)    -   N=6: λ=2nd/3=749.47 nm (Peak)    -   N=7: λ=4nd/7=642 nm (Valley)    -   N=8: λ=nd/2=562 nm (Peak)    -   N=9: λ=4nd/9=499.64 nm (Valley)    -   N=10: λ=4nd/10=449.6 nm (Peak)

As can be seen, and illustrated further in FIGS. 3A and 3B, at leastthree peaks/valleys (N=7-9) occur in the visible spectral range.

If the 7^(th) order valley is used to calculate the change in molecularlayer thickness, when the molecular layer attached to the first layerincreases from 0 nm to 10 nm, the 7^(th) order valley will shift to650.74 nm. Therefore, the ratio between the actual the phase shift ofthe 7^(th) order valley and thickness change equals(650.74-642.40)/10=0.834.

By contrast, if the initial spacing between the two reflecting layers ismade up entirely of the analyte-binding molecules on the end of thefiber, assuming a thickness of this layer of 25 nm, then the first orderpeak will occur at 146 nm, clearly out of the range of the visiblespectrum, so that the device will only see a portion of the regionbetween the O-order valley and the first order peak, but will not seeany peaks, making a shift in the spectral characteristics of theinterference wave difficult to measure accurately.

Not until the total thickness of the reflecting layer approaches about100 nm will the first-order peak appear in the visible spectrum.Assuming a total thickness change of up to 50 nm, the thickness of theoptical element can then be as small as 50 nm, but is preferably on theorder of several hundred nm, so that the phase shift or change inperiodicity of the interference wave can be measured readily by a shiftin the spectral positions of higher-order peaks or valleys, e.g., whereN=3-10.

The ratio between the actual thickness and the measured phase shift isconsidered as a key factor of measurement sensitivity. It can beappreciated how one can adjust the thickness of the optical element andits refractive index to improve and optimize the sensitivity toaccommodate the electronics and optical designs.

FIG. 4 shows an optical assembly 50 that is removably carried on thedistal end of an optical fiber 52 in the assay apparatus. The opticalelement includes a plurality of flexible gripping arms, such as arms 54,that are designed to slide over the end of the fiber and grip the fiberby engagement of an annular rim or detente 56 on the fiber withcomplementary-shaped recesses formed in the arms, as shown. Thisattachment serves to position the optical assembly on the fiber toprovide an air gap 58 between the distal end of the fiber and theconfronting (upper) face of the assembly, of less than 100 nm or greaterthan 2 μm. With an air gap of greater than about 100 nm, but less that 2μm, internal reflection from the upper surface of the optical assemblycan contribute significantly to undesirable fringes that can adverselyimpact the detection accuracy.

With continued reference to FIG. 4, the optical assembly includes afirst optical element 60 similar to optical element 38 described above,and having first and second reflective layers 62, 64, respectively,corresponding to above-described reflective layers 40, 42, respectively.The assembly further includes a second optical element 66 whosethickness is preferably greater than 100 nm, typically at least 200 nm,and whose index of refraction is similar to that of first opticalelement 60. Preferably, the two optical elements are constructed of thesame glass or a polymeric material having an index of refraction ofbetween about 1.4 and 1.6. Layer 64, which is formed of a high index ofrefraction material, and has a thickness preferably less than about 30nm, is sandwiched between the 2 optical elements as shown.

In operation, the optical assembly is placed over the distal fiber endand snapped into place on the fiber. The lower surface of the assemblyis then exposed to a sample of analyte, under conditions that favorbinding of sample analyte to the analyte-binding molecules formingreflective layer 62. As analyte molecules bind to this layer, thethickness of the layer increases, increasing the distance “d” betweenreflective surfaces 62 and 64. This produces a shift in the extrema ofthe interference wave produced by reflection from the two layers, asdescribed above with reference to FIGS. 3A and 3B. This shift in extremaor wavelength, or wavelength period, in turn, is used to determine thechange in thickness at the lower (distal-most) reflecting layer. Afteruse, the optical assembly can be removed and discarded, and replacedwith fresh element for a new assay, for assaying the same or a differentanalyte.

FIG. 5 illustrates an optical assembly and fiber bundle in an embodimentof the invention designed for detecting one or more of a plurality ofanalytes, e.g., different-sequence nucleic acid analytes, in a sample. Afiber bundle 72 is composed of an array, e.g., circular array, forindividual optical fibers, such as fibers 74. The optical assembly,indicated generally at 70, is composed of the basic optical elementsdescribed above with reference to FIG. 4, but in an array format.Specifically, a first optical element 80 in the element provides at itslower distal surface, an array of analyte-reaction regions, such asregions 84, each containing a layer of analyte-binding moleculeseffective to bind to one of the different analytes in the sample. Eachregion forms a first reflective layer in the optical assembly. Onepreferred sensing provides an array of different-sequence nucleic acids,e.g., cDNAs or oligonucleotides, designed to hybridize specifically withdifferent-sequence nucleic acid analyte species in a sample. That is,the array surface forms a “gene chip” for detecting each of a pluralityof different gene sequences.

Also included in the optical assembly are a second optical element 78and a layer 79 of high index of refraction material sandwiched betweenthe two optical elements, and which provides the second reflectingsurface in the optical assembly. The assembly is carried on the fiberbundle 72 by engagement between a pair of flexible support arm, such asarm 76 and an annular rim or detente 86 on the bundle. With the assemblyplaced on the fiber bundle, the lower distal ends of the fibers arespaced from the confronting surface of optical element 78 by an air gap85 whose spacing is preferably less than 100 nm or greater than 2 μm.Further, each of the fibers is aligned with a corresponding assay regionof the optical assembly, so that each fiber is directing light on, andreceiving reflected light from, its aligned detection region. Similarly,the optical coupler in the apparatus, which serves to couple multiplefibers to the detector, preserves the alignment between the arrayregions and corresponding positions on an optical detector, e.g.,two-dimensional CCD. The materials and thickness dimensions of thevarious optical-assembly components are similar to those described abovewith respect to FIG. 4.

The apparatus described in this invention can be used more specificallyfor the following applications:

-   -   (i) with an anti-species antibody carried on the tip, for        screening hybridoma expression lines for cell lines with high        antibody expression;    -   (ii) with an antigen carried on the tip, to characterize high        affinity antibodies against that antigen;    -   (iii) with a protein carried on the tip, for identifying and        characterizing binding partners (DNA, RNA, proteins,        carbohydrates, organic molecules) for that protein;    -   (iv) with a carbohydrate or glycosyl moiety carried on the tip,        for identifying and characterizing binding partners (such as,        e.g., DNA, RNA, proteins, carbohydrates, organic molecules) for        that carbohydrate;    -   (v) with a protein thought to participate in a multi-protein        complex carried on the tip, for characterizing the binding        components and/or kinetics of complex formation;    -   (vi) with a small protein-binding molecule carried on the tip,        for identifying and characterizing protein binders for that        molecule;    -   (vii) with an antibody carried on the tip, for constructing a        calibration curve for the analyte using a set of analytes        standards. Using this calibration curve, one can then determine        the concentration of the analyte in unknown solutions (cell        culture supernatants, biological samples, process mixtures,        etc).    -   (viii) with a single-stranded nucleic acid, e.g., ssDNA or RNA        carried on the tip, for identifying and molecules that bind        specifically to the nucleic acid.

Using a temperature control block, the apparatus and method can also beused to monitor the binding and characterize the binding of animmobilized ssDNA to an oligonucleotide in solution to perform SNPanalysis.

The following examples illustrate various methods and applications ofthe invention, but are in no way intended to limit its scope.

EXAMPLES

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperatures, etc.), but some experimental error anddeviation should, of course, be allowed for.

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of protein chemistry, biochemistry,recombinant DNA techniques and pharmacology, within the skill of theart. Such techniques are explained fully in the literature. See, e.g.,T.E. Creighton, Proteins: Structures and Molecular Properties (W. H.Freeman and Company, 1993); A. L. Lehninger, Biochemistry (WorthPublishers, Inc., current addition); Sambrook, et al., MolecularCloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology(S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington'sPharmaceutical Sciences, 18th Edition (Easton, Pa.: Mack PublishingCompany, 1990); Carey and Sundberg Advanced Organic Chemistry 3^(rd) Ed.(Plenum Press) Vols A and B (1992).

Example 1 Small Molecule-protein Binding Reaction

This example demonstrates the capability to detect the binding ofprotein to small molecule immobilized on a sensor tip and subsequentbindings of multiple antibodies. The two-layer configuration on the tipof an optic fiber is used for this test. The thickness of the firstTa₂O₅ layer is 25 nm and the thickness of the second SiO₂ layer is 770nm. The fiber was purchased from Ocean Optics (Dunedin, Fla.). It wasmanually cut into segments that are 40 mm long. Both ends of thesesegments were polished to standard mirror surface quality. The polishingmethod used here was exactly the same as those for optical lenses andmirrors. One surface of these fiber segments was outsourced to anoptical coating house for Ta₂O₅ layer and SiO₂ layer. This vendoremployed an ion-beam assisted physical vapor deposition (IAPVD) coatermade by Leybold. IAPVD is a commonly used coating technique foranti-reflection and optical filters. The experimental steps included thefollowing (all steps are performed at room temperature unless otherwisenoted):

The fiber tip was coated with a polymer monolayer derivatized withbiotin. The polymer monolayer was prepared using a biotinylated lipid(custom). This lipid was using to form a lipid monolayer on the surfaceof water solution. The monolayer was cross linked using UV light for 15minutes. Clean, dry fibers were then brought in contact with thefloating thin film and the biotin polymer was adsorbed onto the fibertip. The fibers were then dryed at 60° C. for 1 hour. The fiber werethen stored under ambient conditions

The biosensor tip was immersed in 50 μg/ml streptavidin streptavidin(Pierce Biotechnology, Rockford Ill., cat # 21122) in PBS (Invitrogen,Carlsbad, Calif.; cat # 14190078) for 9 minutes and then rinsed brieflywith PBS.

The same tip was dipped into 10 μg/ml rabbit-anti-streptavidin solution(AbCam, Cambridge, Mass.; cat # ab6676-1000) in PBS for 36 minutes andthen washed with PBS briefly.

Finally, the tip was immersed in 50 μg/mL donkey-anti-rabbit antibodysolution antibody (Jackson ImmunoResearch, West Grove, Pa.; cat#711-005-152) in PBS for 25 minutes. A final 10 minute rinse wasperformed in PBS solution.

FIG. 6 shows the real-time response curve for this sequential bindingtest. The vertical axis is the 7^(th) order valley phase shift innanometers. It clearly shows the binding of streptavidin to the biotinalready immobilized on the tip, and subsequent bindings ofanti-streptavidin antibody to streptavidin and a second antibody to thisfirst antibody. The dissociation of the streptavidin layer from the tipwas visible (a small reduction in the optical thickness) at 900 seconds.

Example 2 Biomolecular Interaction Analysis of Kinetics and Affinity ofBiomolecular Interactions

This example illustrates use of the invention to carry out abiomolecular interaction analysis (BIA) measuring kinetics and affinityof biomolecular interactions. The same tip configuration as described inExample 1 was used. The experimental steps included the following (allsteps are performed at room temperature unless otherwise noted):

Mercaptosilane coated tips were prepared using the following procedure.Clean, dry fibers were incubated in a mixture of Toluene: hexanoic acid:mercaptopropyltrioxysilane (10:2:1 volumetric ratio) at room temperaturefor 24 hours. The fibers were rinsed 2× with 10 mL toluene for 5 minuteseach. The fibers were then rinsed 1× with 10 mL of ethanol and driedunder a stream of argon and stored at ambient conditions.

The biosensor tip was first derivatized by immersion in a with 10 μg/mlsolution of rabbit-IgG (Jackson ImmunoResearch, West Grove, Pa.; cat#309-005-003) in PBS for 1 hour.

The coated tip was dipped into 10 μg/ml goat-anti-rabbit antibodysolution (Jackson ImmunoResearch, West Grove, Pa.; cat# 111-005-003) inPBS and remained in it for 15 minutes.

The tip was removed and washed in PBS. To facilitate the dissociation ofthe second antibody from the first antibody, the PBS was agitatedmanually for 20 minutes.

The tip was then dipped into the same goat-anti-rabbit solution again toshow the reproducible association of goat-anti-rabbit to rabbit-IgG.

FIG. 7 shows the on and off curves generated from the association anddissociation of rabbit-IgG and goat-anti-rabbit. The vertical axis isagain the 7^(th) order valley phase shift. The phase shift is directlyrelated to the average thickness with a ratio of 0.834. The ability todetect the on and off curves reliably is essential for measuringinteraction kinetics and affinity.

Example 3 Calculating Affinity Constants from Antibody-antigen Bindingand Release Curves

This experiment demonstrates the calculation of affinity constants frommeasuring on and off curves for two antibodies and their antigen. Theproprietary antibodies were labeled as Ab-1 and Ab-2. The molecularweight of the antigen was about 30 kilodaltons. The same tipconfiguration as described in Example 1 was used. The samemercaptosilane fiber preparation as described in Example 2 was used. Theexperimental steps included (all steps are performed at room temperatureunless otherwise noted):

The fiber tip was activated for covalent attachment of the antigen.Mercaptosilane coated fibers were activated by immersing the sensor tipsin 50 μL of a 50 mg/mL solution of sulfo-SMCC (Pierce Biotechnology,Rockford Ill.; cat # 22322) in DMF (Sigma-Aldrich Chemical Company, StLouis, Mo.; cat # 494488) at for 2 hours. The sensor tips were rinsedbriefly in DMF and dried;

The antigen was covalently bound to the activated fiber tip by immersingthe activated tip in a 20 μg/ml solution of antigen in PBS for 20minutes. The tip was rinsed with PBS for 2 minutes. Following the PBSrinse, the tip was quenched with an aqueous solution of 100 μMethanolamine pH 8.5 (Sigma-Aldrich Chemical Company, St Louis, Mo.; cat# E9508) for 5 minutes and then was rinsed again in PBS for 2 minutes.

The same tip was immersed in antibody for an association test and thereal-time binding data were recorded for 9-15 minutes (depending on theantibody identity and concentration). Once those data were recorded, thetip was again immersed in PBS and agitated to measure the off curve(i.e., dissociation between the immobilized antigen and bound antibody)for 9-15 minutes. The binding (on curve) and dissociation (off curve)measurements were repeated using different concentrations of antibody(25 nM, 150 nM, and 430 nM) and with two different antibodies identifiedas Ab-1 and Ab-2.

FIG. 8 shows the association and dissociation curves at differentconcentrations. The test of 25 nM Ab-2 was not completed because theassociation was extremely slow at this concentration. These illustratedcurves are plots of the raw data.

K_(on), K_(off), and K_(D) were derived from these curves by fitting theraw data with a first order exponential function. By averaging two setsof data, kinetic and affinity coefficients were obtained as follows:

Ab-1 Ab-2 K_(on) = 1.35 × 10⁵ (M⁻¹S⁻¹) K_(on) = 2.01 × 10⁵ (M⁻¹S⁻¹)K_(off) = 5.55 × 10⁻⁵ (S⁻¹) K_(off) = 8.15 × 10⁻⁵ (S⁻¹) K_(D) =K_(off)/K_(on) = 3.99 × 10⁻⁹ (M) K_(D) = K_(off)/K_(on) = 4.45 × 10⁻⁹(M)

Example 4 NHS-ester Activated Tips

The same tip configuration as described in Example 1 was used. The samemercaptosilane fiber preparation as described in Example 2 was used.Mercaptosilane coated fibers were activated by immersing the sensor tipsin 50 μL of a 50 mg/mL solution of sulfo-SMCC (Pierce Biotechnology,Rockford Ill.; cat # 22322) in DMF (Sigma-Aldrich Chemical Company, StLouis, Mo.; cat # 494488) at for 2 hours. The sensor tips were rinsedbriefly in DMF and dried.

Amine containing molecules can be covalently bound to this surfacethrough formation of a stable amide linkage. Molecules that do notcontain free amines are not immobilized through the NHS moiety, butthese molecules can still bind to the surface through non-specificbinding. This non-specific binding can be multi-layered whereas thecovalent immobilization through the NHS esters will be in a single layercontrolled by the availability and accessibility of the NHS ester.

In this set of experiments, a bis amino PEG (MW 3300) (ShearwaterPolymers, San Carlos, Calif.) was used as a test compound to covalentlybind to the activated surface. A PEG (MW 8000) (Sigma-Aldrich ChemicalCompany, St Louis, Mo.; cat # 04162) that contained no free amino groupswas used as a negative control. This negative control was used to lookfor any non-specific or multi-layered binding that might be inherent toPEG polymers on this surface.

FIG. 9 shows the time course of the treatment of the activatedmercaptosilane tip with the test molecules. The activated tip showed adistinct increase in optical thickness upon exposure to the 0.1 mg/mLbis amino PEG (MW 3300) in PBS. This increase is stopped when the bisamino PEG solution is replaced by the PBS buffer. The activated tipexposed to 0.1 mg/mL PEG (MW 8000) in PBS, which contains no amines,shows a small initial increase in optical thickness but the tracequickly becomes flat. From this it can be concluded that the PEG polymerdoes not have intrinsic non-specific binding and that the binding seenfor the bis amino PEG is attributed to the specific covalentimmobilization of the amine group.

Example 5 Antibody Derivatized Tips Using NHS-ester Chemistry

This example illustrates the binding of a low molecular weight moleculebinding to an immobilized high molecular weight molecule. Using the sameNHS ester terminated surface described in Example 4 and the same tipconfiguration as described in Example 1, an anti-biotin antibody wasimmobilized to 3 fibers. Immobilization of the antibody was accomplishedby immersing the activated fiber in a 20 μg/mL solution of mouseanti-biotin antibody (Biodesign, Saco Minn.; cat # H61504M) in PBS for 1hour at room temperature. The tip was rinsed with PBS for 2 minutes.Following the PBS rinse, the tip was quenched with an aqueous solutionof 100 μM ethanolamine pH 8.5 (Sigma-Aldrich Chemical Company, St Louis,Mo.; cat # E9508) for 5 minutes and then was rinsed again in PBS for 2minutes.

The first fiber was exposed to a solution of 200 μg/mL biotin (PierceBiotechnology, Rockford Ill.; cat # 29129) in PBS. Controls using asolution of sucrose (Sigma-Aldrich Chemical Company, St Louis, Mo.; cat# S8501) (2 mg/mL) and PBS were carried out on the second and the thirdfibers to determine baseline noise. Data from these tests are shown inFIG. 10. Biotin binding is seen as an increase in optical thickness,whereas exposure to sucrose shows no detectable increase over baseline(PBS).

Another negative control was carried out using an irrelevant antibody(anti-Lewis Y antibody from Calbiochem, San Diego Calif.; cat# 434636)immobilized in an identical fashion to the anti-biotin antibody above.This immobilized antibody was exposed to a solution of 200 μg/mL biotin.The lack of biotin binding to this antibody indicates that the biotinbinding to the anti-biotin antibody is a result of specific interactionsand not due to non-specific binding.

While the invention has been particularly shown and described withreference to a preferred embodiment and various alternate embodiments,it will be understood by persons skilled in the relevant art thatvarious changes in form and details can be made therein withoutdeparting from the spirit and scope of the invention.

All references, issued patents and patent applications cited within thebody of the instant specification are hereby incorporated by referencein their entirety, for all purposes.

1. An assembly for use in detecting an analyte in a sample based oninterference, comprising: an optical fiber having a tip; a first opticalelement adapted for coupling to a light source through a mechanicalcoupling that engages the first optical element with the fiber andprovides an air gap between the first optical element and the fiber; andsecond optical element attached to the first optical element, the secondoptical element commensurate in size with the fiber tip and adapted forcoupling to the first optical element, said second optical elementcomprising a transparent material, a first reflecting surface, and asecond reflecting surface separated from the first reflecting surface bythe transparent material, said first and second reflecting surfacesseparated by at least 50 nm, wherein said first reflecting surfacecomprises a layer of analyte binding molecules, and an interferencebetween light reflected into the fiber from said first and secondreflecting surfaces varies as analyte in the sample binds to the analytebinding molecules.
 2. The assembly of claim 1, wherein said secondreflecting surface comprises a layer of material having refractive indexgreater than the refractive index of said first optical elementtransparent material.
 3. The assembly of claim 1, wherein the separationbetween said first and second reflecting surfaces is between 100 nm and5,000 nm.
 4. The assembly of claim 3, wherein the separation betweensaid first and second reflecting surfaces is between 400 nm and 1,000nm.
 5. The assembly of claim 1, wherein the refractive index of saidfirst optical element transparent material is less than 1.8.
 6. Theassembly of claim 5, wherein said first optical element transparentmaterial is a material selected from the group consisting of SiO₂ and atransparent polymer.
 7. The assembly of claim 6, wherein saidtransparent polymer comprises polystyrene or polyethylene.
 8. Theassembly of claim 1, wherein the second reflecting surface comprises alayer of material having a refractive index greater than 1.8.
 9. Theassembly of claim 8, wherein said second reflecting surface layercomprises Ta₂O₅.
 10. The assembly of claim 9, wherein the thickness ofsaid second reflecting surface layer is between 5 nm and 50 nm.
 11. Theassembly of claim 1, wherein said layer of analyte binding moleculescomprises a molecule selected from the group consisting of a protein, asmall molecule, a nucleic acid and a carbohydrate.
 12. The assembly ofclaim 11, wherein said protein is selected from the group consisting ofan avidin, a streptavidin, an antibody, and an antibody fragment. 13.The assembly of claim 1, wherein the thickness of said first opticalelement is greater than 100 nm.
 14. The assembly of claim 13, whereinthe thickness of said first optical element is greater than 200 nm. 15.The assembly of claim 1, wherein said air gap is less than 100 nm. 16.The assembly of claim 1, wherein said air gap is greater than 2 μm. 17.A two dimensional array of the assemblies of claim
 1. 18. An apparatusfor detecting an analyte, comprising: the two dimensional array of theassemblies of claim 17; a light source for directing light onto saidfirst and said second reflecting surfaces; and a detector that receiveslight from said first and said second reflecting surfaces and detects achange in optical thickness of said first reflecting surface uponexposure of said first reflecting surface to said analyte.
 19. A methodfor detecting analyte in a sample, comprising: providing the apparatusof claim 18 and a sample; exposing said first reflecting surface to saidsample, and determining whether said exposure results in a change inoptical thickness of said first reflecting surface.
 20. An apparatus fordetecting an analyte, comprising: the assembly of claim 1; a lightsource for directing light onto said first and said second reflectingsurfaces; and a detector that receives light from said first and saidsecond reflecting surfaces and detects a change in optical thickness ofsaid first reflecting surface upon exposure of said first reflectingsurface to said analyte.
 21. A method for detecting analyte in a sample,comprising: providing the apparatus of claim 20 and a sample; exposingsaid first reflecting surface to said sample, and determining whethersaid exposure results in a change in optical thickness of said firstreflecting surface.
 22. A kit, comprising: an assembly comprising anoptical fiber having a tip; first optical element adapted for couplingto a light source through a mechanical coupling that engages the firstoptical element with the fiber and provides an air gap between the firstoptical element and the fiber; a second optical element attached to thefirst optical element, the second optical element commensurate in sizewith the fiber tip and adapted for coupling to the first opticalelement, said second optical element comprising a transparent material,a first reflecting surface, and a second reflecting surface separatedfrom the first reflecting surface by the transparent material, saidfirst reflecting surface and said second reflecting surface separated byat least 50 nm, wherein said first reflecting surface binds a layer ofanalyte binding molecules and an interference between light reflectedinto the fiber from said first and second reflecting surfaces varies asanalyte binds to the analyte binding molecules, and said secondreflecting surface comprises a layer of material having an index ofrefraction greater than the refractive index of said optical elementtransparent material; and instructions for binding said layer of analytebinding molecules to said first reflecting surface.
 23. The kit of claim22, further comprising a reagent for chemically modifying said firstreflecting surface and instructions for using said reagent.